Tryptic Hydrolysis of Dodecapeptides Possessing Paired Basic Residues

Four dodecapeptides of general formula Tyr-Gly-Gly-Phe-Met-X-X-Tyr-Gly-Gly-Phe-Met-NH₂ (Enk-X-X-Enk-NH₂) possessing X = Arg or Lys have been synthesized and subjected to cleavage by trypsin. The peptide with the sequence containing -Lys-Arg-, depicted as BI-NH₂, represents the 100–111 segment of proenkephalin. The time course of the degradation was followed by high performance liquid chromatography. This method allows one to observe the formation of not only the final but also intermediate peptides. Among the peptides studied, the most susceptible to the cleavage was BI-NH₂. The primary hydrolysis proceeded rapidly at the arginine residue, followed by slow release of arginine. The other peptides (with -Arg-Arg-, -Lys-Lys- and -Arg-Lys-) were cleaved at both possible positions, but the resulting mixture contained Enk-X as a major product, which was the result of both primary and secondary cleavage.     

Hydrolysis of DNA by a Branched Peptide without Aromatic Residues

A branched peptide Nα, Nɛ-di (l-leucyl)-l-lysine was found to efficiently cleave supercoiled double-strand DNA such as PUC19 DNA at optimum pH 4.0 in 40 mmol/l Britton–Robbinson buffer. The T4 ligase experiment implied that the DNA cleavage occurs via a hydrolytic path. The dependence of the cleavage reaction on the ionic strength indicated that the interaction of DNA with the branched peptide involve only electrostatic binding.     

Degradation Sequence of Glycinin by Tryptic Hydrolysis

Native glycinin was split by trypsin in limited regions under high ionic strength condition and converted into glycinin-T. Initially, the intermediary subunits of glycinin converted into fragments of 48,000 (IST-T) and 44,500 (IST-2) molecular weights. These fragments were transitory intermediates and then they were cleaved yielding fragments of 32,500 (IST-3) and 29,000 (IST-4) molecular weights. Analyses of the mole fractions of the fragments revealed that IST-2 was rapidly split into IST-4 followed by splitting of IST-1 into IST-3.

Subsequently, glycinin-T was dissociated with 6 m urea and subjected to gel filtration and ion exchange chromatography in order to isolate IST-3 and IST-4. Acidic-urea gel electrophoresis of the isolated 1ST showed that IST-3 contained B₁ B₂ and B₃ subunits, whereas IST-4 contained B₄ subunit. These results indicated that IST-3 was formed from IS—1 and IS-2, and IST-4 from IS-3.     

Effect of Dimethylsulfoxide on Hydrolysis of Lipase

To establish an industrially feasible reaction process, the effect of dimethylsulfoxide (DMSO) added to an aqueous solution on the hydrolysis of lipase was investigated using fluorescent substrates. Several lipases from microorganisms were improved in their hydrolysis activities against 4-methylumbelliferyl oleate by DMSO. Variation was found in the effect of DMSO depending on the species of lipase. After the high stability of the lipase from Pseudomonas fluorescens in DMSO solution was confirmed, hydrolysis by this lipase of four acyl-4-methylumbelliferones was studied kinetically at different DMSO concentrations. DMSO added to an aqueous solution increased the V_max of this lipase for a substrate with strong hydrophobicity, and decreased that value for a substrate with an opposite property. On the other hand, DMSO had a very small effect on K_m for each substrate. A fluorometric study suggested that DMSO induced a change of the chemical environment that surrounded tryptophan residues of the lipase. Such conformational change would be one of the causes of the DMSO-induced alteration of its reactive property. These results suggest that the addition of DMSO may be a novel method of ‘solvent engineering’ of this enzyme.     

Tryptic hydrolysis of hGH-RH(1-29)-NH2 analogues containing Lys or Orn in positions 12 and 21.

Two analogues of the 29 amino acid sequence of human growth hormone-releasing hormone, namely [Nle27]hGH-RH(1-29)-NH2 and [Orn(12,21),Nle27]hGH-RH(1-29)-NH2, have been synthesized and subjected to digestion by trypsin. The course of degradation was followed using RP-HPLC and ESI-MS. Several intermediates and final products of degradation were identified and conclusions regarding the rate of cleavages at different positions occupied by Lys and Arg residues were drawn. The analogue containing ornithine was found to be less susceptible to hydrolysis by trypsin: the 12-13 and 21-22 peptide bonds were completely resistant to the cleavage. The results show that by replacing Lys with Orn, a possibility exists to design new peptides, which could be more stable in biological fluids.     

Expression of monomeric insulin precursor in Pichia pastoris and its conversion into monomeric B27 Lys destripeptide insulin by tryptic hydrolysis

Monomeric B27 Lys destripeptide insulin (B27 Lys DTrI) was designed and produced from its precursor expressed in Pichia pastoris through tryptic hydrolysis instead of the less efficient tryptic transpeptidation. The monomeric B27 Lys DTrI precursor (MIP) was purified from a cultured medium of P. pastoris by a combination of hydrophobic, size-exclusion, and ion-exchange chromatography. The purified MIP was converted, by tryptic hydrolysis, to B27 Lys DTrI, which was then purified by ion-exchange chromatography to homogeneity as assessed by native gel electrophoresis, HPLC, amino acid composition,and electrospray mass-spectrometric analysis. B27 Lys DTrI exhibited superior monomeric properties in size-exclusion chromatography. The yield of MIP was 200 mg per liter of culture, and the overall yield of purified B27 Lys DTrI from the crude MIP was 70%. The in vivo biological activity of B27 Lys DTrI as determined by the mouse convulsion assay was 21 U/mg, identical to that obtained by semisynthesis.     

蛋白质的酶水解过程研究

进行了蛋白质酶水解过程的研究。结果表明木瓜蛋白酶对混合蛋白质的亲和力最强 ,而 1398蛋白酶的亲和力最弱。也表明作用位点和亲和力之间有一定的对应关系 ,Km值和作用位点氨基酸含量比例的相关系数为 0 .90 9。温度影响结果表明温度较低时温度升高加速水解反应过程处主要地位 ;当温度较高时 ,酶失活过程处主导地位。在一定水解时间内的讨论最适温度条件具有更明确的针对性 ,从本研究的采用胰酶 (胰蛋白酶和胰凝乳蛋白酶 )水解 4h的条件下 ,反应温度控制在45～ 5 0℃之间最适     

Pretreatment and Hydrolysis of Brewer's Spent Grains

Brewer's spent grains (BSG), the voluminous residue after mashing, contains on dry weight basis about 40–50 % polysaccharides. For the recovery of soluble carbohydrates from BSG different physical, thermal and enzymatic treatments were used to solubilize the polysaccharides in BSG. Heating by microwave radiation to 160 °C in the presence of 0.1 M HCl released 35 % of the material in the form of reducing sugar, indicating that about 80 % of the polysaccharides were hydrolyzed. Nevertheless, 0.1 M acetic acid will even solubilize 30 % of the material as oligosaccharides on a prolonged incubation when pretreating the spent grains by extrusion cooking. A combination of the enzymes Ceremix Plus MG and CelluPract AL 70 achieved an almost 25 % release of saccharides after 4 hrs of incubation at 50 °C. A combination of extrusion cooking and enzymatic hydrolysis seems to be a very promising procedure.     

Isolation of a tripeptide (Ala-Gly-Ser) exhibiting weak acetylthiocholine hydrolyzing activity from a high-salt soluble form of monkey diaphragm acetylcholinesterase

A high-salt soluble form of acetylcholinesterase (AChE) was purified from monkey (Macaca radiata) whole diaphragm by a two step affinity chromatographic procedure using m-aminophenyl trimethylammoniumchloride hydrochloride-Sepharose and procainamide-Sepharose columns. The purified enzyme showed three major protein bands at 80 kDa, 78 kDa and 60 kDa on SDS-gel electrophoresis. [³H]Diisopropyl fluorophosphate ([³H]DFP) labeled enzyme also gave three radioactive peaks corresponding to these three bands. The purified enzyme pretreated with dithiothreitol and subjected to limited trypsin digestion gave a peptide fragment of molecular weight 300 Da showing weak acetylthiocholine hydrolyzing activity as identified by Sephadex G-25 gel filtration. Sequence analysis showed that the active peptide fragment was a tripeptide with the sequence Ala-Gly-Ser. When the purified AChE was labeled with [³H]DFP, digested with trypsin and subjected to Sephadex G-25 chromatography, a radioactive peak that would correspond to the tripeptide fragment was seen. The kinetics, inhibition characteristics and binding characteristics to lectins of the active peptide fragment was compared with the parent enzyme.A synthetic peptide of sequence Ala-Gly-Ser was also found to exhibit acetylthiocholine hydrolyzing activity. The kinetics and inhibition characteristics of the synthetic peptide was similar to those of the peptide derived from the purified enzyme, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 29480 times less than that of the purified AChE.Abbreviations AChE Acetylcholinesterase - BW284C51 1,5-bis(4-allyl dimethylammonium phenyl) pentan 3-one-dibromide - DFP Diisobropyl fluorophosphate - TIPP Tetra isopropyl pyrophosphoramide - TPCK N-Tosyl-L-phenylalanylchloromethyl ketone - MAP m-Aminophenyl trimethylammonium chloride - RCA₁ Ricinus communis agglutinin 120 - TEAB Tetraethylammonium bromide - DTT Dithiothreitol     

Hydrolysis of liquefied corn starch in a membrane reactor

The objective of this study was to develop a continuous hydrolysis process for the enzymatic saccharification of liquefied corn starch using a membrane reactor. A residence time distribution study confirmed that the membrane reactor could be modeled as a simple continuous stirred tank reactor (CSTR). Kinetic studies indicated that the continuous reactor operated in the first-order region with respect to substrate concentration at substrate concentrations greater than 200 g/L. At a residence time of 1 h and an enzyme concentration of 1 g/L, the maximum reaction velocity (V(m)) was 3.86 g glucose/L min and the apparent Michaelis constant (K(m) (')) was 562 g/L. The K(m) (') value for the continuous reactor was 2-7 times greater than that obtained in a batch reactor.Kinetic data were fit to a model based on the Michaelis-Menten rate expression and the design equation for a CSTR. Application of the model at low reactor space times was successful. At space times of 6 min or less, the model predicted the reactor's performance reasonably well. Additional work on the detection and quantitation of reversion products formed by glucoamylase is required. Isolation, detection, and quantitation of reversion products by HPLC was difficult. Detailed analysis on the formation of these reversion products could lead to better reactor designs in the future.     

Tryptic digestion of the (Na + K)-ATPase is both sensitive to and modifies K+ interactions with the enzyme

Tryptic digestion of the (Na + K)-ATPase in the presence of choline chloride or NaCl (Na-type) and in the presence of KCl (K-type) produced distinct patterns of peptide fragments and losses of catalytic activity. TheK _0.5 for K⁺ to shift digestion from the Na-type, and its sensitivity to dimethyl sulfoxide and Triton X-100, were consistent with K⁺ acting at sites on the cytoplasmic face of the enzyme through which the K-phosphatase reaction also is activated. Reagents favoring the E₁ conformational states, oligomycin, Triton, and ATP, shifted the pattern toward the Na-type, whereas those favoring E₂ states, dimethyl sulfoxide, MgCl₂, and MnCl₂, shifted the pattern toward the K-type. Na-type digestion caused a greater loss of K-phosphatase than (Na + K)-ATPase activity, and the residual K-phosphatase activity was more sensitive to inhibition by Triton and ATP but stimulated more by dimethyl sulfoxide and inhibited less by P_i and MnCl₂; all these effects are consistent with such digestion shifting equilibria toward E₁ enzyme states. Accordingly, theK _0.5 for K⁺ to activate the (Na + K)-ATPase was increased. However, theK _0.5 for the K-phosphatase was unchanged; this observation requires revision of previous formulations, and bears on additional aspects of enzyme activity as well.     

工业废羊毛酸解工艺条件选择

毛发蛋白盐酸水解提取胱氨酸时 ,温度、毛酸比、时间等因素影响到毛发蛋白的水解率和产品胱氨酸的总收率。通过正交试验法 ,进行了废羊毛酸解工艺参数的试验研究 ,确定了最佳酸解工艺参数为 :温度 1 0 5℃ ,毛酸比(W/V) 1∶1 .7,连续水解时间 7.0h。     

Isolation and Identification of Large Overlapping Fragments of Rabbit Myelin Basic Protein Produced by Limited Peptic Hydrolysis

Treatment of rabbit myelin basic protein component 1 with pepsin (enzyme:substrate, 1:500 w/w) in 0.5 M-ammonium formate (pH 6.00) for 15-20 min at room temperature resulted in limited cleavage of the protein. The resulting fragments were isolated by ion-exchange chromatography and gel filtration and identified by amino acid and COOH-terminal analyses and by tryptic peptide mapping. All of the possible products resulting from incomplete cleavages at the highly susceptible Phe44-Phe45, Phe87-Phe88, Leu109-Ser110, and Leu151-Phe152 bonds were isolated: peptides (1-151), (1-109), (1-87), (45-168), (45-151), (45-109), (88-168), (88-151), and (110-168). Of these, peptides (1-151), (1-87), and (88-151) were recovered in the greatest yield (0.14-0.19 mol per mol of starting protein). Relatively low yields (0.04 mol/mol starting protein) were obtained for peptides (1-109) and (110-168), indicating that the Leu109-Ser110 bond is somewhat more resistant to peptic cleavage than are the Phe-Phe and Leu-Phe bonds. Smaller fragments of the basic protein were also recovered: peptides (1-44), (1-28), (45-87), (88-109), (110-151), and (152-168). Many of the individual peptides could be readily identified in electrophoretograms of the total peptic digest. The relative electrophoretic mobilities of the above-mentioned peptides, together with the previously isolated peptides (1-14) and (15-44), were determined in 15% (w/w) polyacrylamide slab gels containing 1 M-acetic acid and 8 M-urea.     

不同纤维素原料超临界水解的研究

分别以稻草秸秆、经预处理的稻草秸秆、脱脂棉、微晶纤维素和定性滤纸为原材料,利用间歇式的超临界反应设备,在400℃的盐浴中进行木质纤维素的超临界水解,采用3,5-二硝基水杨酸(DNS)法对产物中的还原糖进行测定,研究反应时间对不同纤维素原料水解产糖的影响。结果表明:在超临界条件下,不同原料在较短的时间内还原糖含量均出现峰值,随着反应时间的延长还原糖产量呈现下降的趋势;稻秆、预处理后的稻秆、脱脂棉、微晶纤维素和定性滤纸的最大产糖量分别为7.42、9.05、12.55、18.01和14.24 g/L;与此对应的最佳反应时间分别为3.5、4、3、3、4 min;对应的最大还原糖产率分别为14.84%、18.10%、25.10%、36.02%、28.48%。     

Enzymatically Enantioselective Hydrolysis of Prochiral 1,3-Diacyloxyglycerol Derivatives

An enzymatically enantioselective ester hydrolysis of prochiral 1,3-diacyloxy-2-substituted-2-propanol to chiral 1-acyloxy-2,3-propanediol was studied. The (R)-monoester was prepared by selection of a suitable lipase and alkyl chain length of the substrate diester. Lipase D from Rhizopus delemer gave (R)-1-isobutyryloxy-2-(2,4-difluorophenyl)-2,3-propanediol with 97%ee and 87% yield at 15°C and pH 5.5. The (R)-monoester is a key intermediate of azole antifungal agents.     

木瓜蛋白酶水解壳聚糖的工艺研究

本文通过正交试验对木瓜蛋白酶水解壳聚糖的工艺进行优化,并对降解过程中粘度、还原糖等一些指标的动态变化进行研究。结果显示,木瓜蛋白酶在反应温度45℃下降解壳聚糖的最佳工艺条件为壳聚糖的脱乙酰度70%,pH4.0,底物浓度1%,壳聚糖与酶的比例为25∶1(w/w)。其中底物脱乙酰度对酶解效果影响呈极显著水平,pH值影响呈显著水平。木瓜蛋白酶可较为有效地降解脱乙酰度为70%的壳聚糖,在其最适条件下对壳聚糖水解约60min,可控制产物平均分子量在1万以内。木瓜蛋白酶起始降解速率很快,20min后VDP变化趋于平稳,60min后基本维持在93～94%上下。反应进行60min后产物的平均分子量约为9000。还原糖含量在反应进行150min之后,还原糖的生成速度基本趋于平稳。     

牦牛骨蛋白的酶解条件研究

以蛋白质水解度为评价指标,辅以固形物溶出率,比较了中性蛋白酶、菠萝蛋白酶和木瓜蛋白酶对牦牛骨蛋白的水解效果,研究了酶用量、料液比(底物浓度)、酶解时间对水解度的影响,采用正交试验对酶解条件进行了优化。结果显示,木瓜蛋白酶是牦牛骨蛋白水解的适宜催化剂。在一定条件下,样品水解度随酶用量和酶解时间的增加而增大,底物浓度过低或过高均不利于原料中蛋白质的酶解。木瓜蛋白酶水解牦牛骨蛋白最佳条件为:酶解温度60℃,酶解时间8 h,酶用量3500 U/g蛋白质,料液比1:25(g:m l)。     

Hydrolysis of plant seed gums by microwave irradiation

Under microwave irradiation (MW), the seed gums, guar and Ipomoea quamoclit were hydrolyzed to constituent monosaccharides and oligosaccharides in very mild conditions and short reaction time. Under MW both the seed gums could be completely hydrolyzed using very dilute acid (0.00625N H₂SO₄) within two minutes. Hydrolysis occurs in 2 min and 20 s even in absence of acid under the MW irradiation. Thus hydrolytic fragmentation under MW provides an efficient tool in structural elucidation of polysaccharides.     

直接加热膨化蔗渣酶法水解的研究

采用加热时间为２０ｍｉｎ,压力为２．０ＭＰａ,温度为１２０℃的直接加热膨化甘蔗渣为水解底物、日本ｙｓｋｕｌｔ生物化学试剂公司生产的ｏｎｏｚｕｋＲＳ型纤维纯洁酶粉进行蔗渣的酶法水解实验,考察了蔗渣中纤维素的酶解还原糖得率与反应时间、酶浓度、ｐＨ值、缓冲液种类、离子强度以及固液比的关系,结果表明：当固液比为５％（ｗ／ｖ）,酶浓度＞１．２０ｍｇ／ｍｌ时,还原糖得率随酶浓度的增加变化不显著;本实验条件下,缓冲液种类和离子强度对还原糖得率几乎没有影响;水解最适宜ｐＨ值为４．２～４．９;最佳反应温度为５０℃。     

响应曲面法制备蛹虫草子实体酶解液的研究

为了将蛹虫草开发成为便于人们食用的产品形式,本实验以不同的酶对蛹虫草进行水解得到蛹虫草酶解液.以水解度和酶解液中腺苷含量为目标,确定选用木瓜蛋白酶.以水解度为响应指标,应用响应曲面法对蛹虫草酶解条件进行优化,根据Box-Behnken中心组合实验设计原理,选取酶解温度、酶解时间、加酶量三因素三水平进行中心组合实验,响应曲面分析结果表明水解最佳条件为:酶解温度60.92℃,酶解时间11.85 h,加酶量1.02％,此条件下蛹虫草的水解度达到最大.水解度验证值61.27％与预测值60.76％接近,说明建立模型正确.